Inactivating organisms using carbon dioxide at or near its supercritical pressure and temperature conditions

ABSTRACT

Whole organisms are inactivated by at least a factor of 10 6  using carbon dioxide at or near its supercritical pressure and temperature conditions.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to whole organisms which have beeninactivated by at least 10⁶ using carbon dioxide at or near itssupercritical pressure and temperature conditions, immunogeniccompositions thereof, methods for preparation, and methods forimmunization.

2. Description of the Related Art

Vaccines represent one of the seminal developments in our ongoing battleagainst disease. Vaccination is still the best defense against existing,novel, and manipulated pathogens. The earliest whole-cell vaccines wereprepared by inactivating a given pathogen using chemical or heatprocesses. Whole-cell vaccines have significant advantages overattenuated and subunit vaccines. Chemical or thermal inactivation of thepathogen is simple and inexpensive, and provides rapid access to avaccine. Both subunit vaccines and attenuated vaccines requireconsiderable time and expense before they can be put to use. Despite theadvantages of chemical inactivation, chemically inactivated vaccinessometimes fail to elicit robust and protective immune responses [2-4].The addition of adjuvants to these preparations may boost the immuneresponse, but immunity is still insufficient in many cases and mayrequire frequent boosting.

Many complications associated with chemically inactivated vaccines arisefrom the simple fact that inactivation alters the chemical properties ofkey antigens required to elicit a protective immune response. Thedevelopment of a rapid, inexpensive, and effective process to inactivatea pathogen while maintaining the integrity of its antigens wouldrepresent a powerful new tool in vaccine development.

Recent work has effectively demonstrated that microbes inactivated by anon-denaturing process do, in fact, elicit more robust immune responsesthan chemically inactivated pathogens [5]. “Ghosts” as they are knowncolloquially, are the empty shells of microbes that have beeninactivated by the controlled expression of the PhiX174 lysis gene “E”[6]. Essentially the cytoplasmic contents of the cells are expelled viathe transmembrane tunnel formed by the lysis protein [6]. Vaccinesprepared through this genetic manipulation have been shown to besuperior to chemically inactivated pathogens, most likely due to thenon-denaturing inactivation procedure [3]. Moreover, it is hypothesizedthat the more robust immune response is not simply a function ofindividual proteins, but also is related to the route of antigenpresentation.

Cell walls remain largely intact, native surface antigens are preserved,and bioadhesive properties are likely maintained in ghost vaccinepreparations. All of these characteristics endow ghost vaccines withinherent adjuvant properties that contribute to protective immuneresponses [3, 7-17]. The usefulness of the bacterial ghost system isextended by inactivating bacteria expressing antigens that are derivedfrom other pathogens. The end result is a vaccine with inherent adjuvantproperties that is protective against any number of desired bacterial,viral, protozoan, and fungal pathogens [12, 13, 15, 16, 18]. There areconcerns about the endotoxicity of lipid A and lipopolysaccharide (LPS)in these whole cell vaccines. However, it has been demonstrated thatendotoxicity is not a real limit to the use of ghost vaccines [17].

Despite its promise, the ghost vaccine technology exhibits a number ofdrawbacks. The first of these concerns centers on safety. The phagelytic system employed typically results in only a 4-log reduction incolony forming units (CFU) [5]. The remaining organisms must beinactivated by further processing. This may or may not be the case. Theghost system uses an additional kill mechanism to inactivate theremaining survivors [5]. This layering of genetic systems in the ghosttechnology is a cause for additional concern. Because these geneticsystems are maintained within the chosen cells by selection on variousantibiotic containing media [19, 20], lateral transfer of antibioticresistance to other pathogens within an individual is a possibility[21].

In addition to safety concerns, the ghost system only works withGram-negative bacteria. Furthermore, genetic manipulation of additionalserotypes may be required to generate a broadly protective vaccine.Therefore, the applicability of the ghost technology is limited to thegram-negative bacteria that are tractable to genetic manipulation. Theselimitations preclude a significant number of pathogens, notably:Staphylococcus aureus, Staphylococcus epidermidis, Streptococcuspneumoniae, Streptococcus agalactiae, Streptococcus pyrogenes,Enterococcus spp., Bacillus anthracis, Bacillus cereus, Lactobacillusspp., Listeria monocytogenes, Nocardia spp., Rhodococcus equi,Erysipelothrix rhusiopathiae, Corynebacterium diptheriae,Propionibacterium acnes, Actinomyces spp., Clostridium botulinum,Clostridium difficile, Clostridium perfringens, Clostridium tetani, andPeptostreptococcus spp. The applicability of ghost vaccine technologiesis further limited by its failure to inactivate spores, which areinsensitive to induction of lysis genes due to their dormant nature.

Whole-cell vaccines produced on the ghost vaccine technology aresuperior to chemically inactivated pathogens, but cannot be developedrapidly. Even if the microbe is previously known, considerable time andexpense are required to generate a new ghost vaccine for a givenpathogen, especially for novel or genetically intractable pathogens. Thepresent invention does not require introduction of a phage lysis geneand induction of a lytic program.

The need for new and broadly applicable inactivation technologies isexacerbated by the very nature of biological weapons. The pathogens thatare, or may be employed as bio-warfare and bio-terror agents such asAnthrax, Tularemia, Botulism, Plague, Epsilon toxin, Q fever,enterotoxin B, Typhus fever, Melioidosis, and Brucellosis are notusually endemic diseases in humans. As such there is very little, ifany, commercial advantage to generating vaccines using expensive andtime-consuming techniques. An appealing alternative that would speed theproduction of such vaccines and enable quick response to emergingserotypes is an inactivation technology that in and of itself generateshigh quality vaccines. Bacterial inactivation by supercritical CO₂represents such a technology. The technology for using supercritical CO₂is well-known and has been adapted to large industrial applications,including the extraction of natural compounds from plant materials [22]and detoxification of contaminated soil [23]. Supercritical CO₂applications have also found their way into medical circles as a processfor bone de-lipidation [24], drug manufacture [25], and sterilizationamong others [1]. The first attempts to use supercritical CO₂ as asterilant resulted in inadequate levels of inactivation [26].

Recently, in U.S. Pat. No. 6,149,864 to Dillow et al. (the entirecontent of which is expressly incorporated hereinto by reference), theuse of supercritical CO₂ was disclosed as an alternative to existingtechnologies for sterilizing a wide range of products for the healthcareindustry with little or no adverse effects on the material treated.Specifically, the Dillow '864 patent disclosed the inactivation of awide range of vegetative microbial cells using supercritical carbondioxide with agitation and pressure cycling. However, only onespore-forming bacterium was investigated in the Dillow '864 patent,specifically, B. cereus. No disclosure appears in Dillow '864 patentregarding the efficacy of the therein suggested techniques usingcurrently accepted bio-indicator standards used to judge sterilization(i.e., B. stearothermophilus and B. subtilis). Subsequently, however,other investigators achieved only a 3.5-log reduction in B. subtilisspore forms using the process disclosed in the Dillow '864 patent [27].

In addition to bacterial inactivation, viral inactivation is realizedusing supercritical CO₂ [28]. Moreover it has been shown thatsterilization by supercritical CO₂ does not affect the properties of abiodegradable polymer (PLGA) and leaves bacterial cells intact [1].

It would therefore be desirable if processes could be provided wherebyorganisms are inactivated utilizing near or supercritical CO₂ for thepurpose of generating whole-cell therapeutic agents. It is towardsfulfilling such a need that the present invention is directed.

SUMMARY OF THE INVENTION

In general, the methods of the present invention result inwhole-organism therapeutic agents by treatment of the organisms usingnear or supercritical carbon dioxide. In preferred embodiments, methodsof this invention treat organisms with near or supercritical carbondioxide at pressures between about 1000 psi to about 3500 psi, attemperatures in the range of between about 25° C. to about 60° C., andtimes ranging from about 10 minutes to about 12 hours. In especiallypreferred embodiments, the present invention utilizes the techniquesdisclosed in commonly owned Int'l Patent Application Serial No.PCT/US2004/020152, filed on Jun. 17, 2004, the entire content of whichis expressly incorporated hereinto by reference.

Other objects and advantages of the present invention will becomeapparent from the following detailed description when viewed inconjunction with the accompanying drawings, which set forth certainembodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a presently preferred apparatus used forinactivation;

FIG. 2 is a detailed schematic view of the pressure vessel employed inthe apparatus of FIG. 1; and

FIG. 3 shows inactivation of whole cells: scanning electron microscopy(SEM) shows untreated bacteria (FIGS. 3A-3C) and treated bacteria (FIGS.3D-3F) have intact cell walls, protein extracts of untreated and treatedbacteria were separated by molecular weight (cf. standards in markerlane) using one-dimensional polyacrylamide gel electrophoresis (PAGE)under denaturing conditions (FIG. 3G) and shows that total protein wassubstantially unchanged, and two-dimensional gel electrophoresis athigher resolution with isoelectric focusing under native conditions andseparation by molecular weight under denaturing conditions for untreatedbacteria (FIG. 3H) or treated bacteria (FIG. 3I) shows that proteins aresubstantially not denatured or lost by inactivation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The detailed embodiment of the present invention is disclosed herein. Itshould be understood, however, that the disclosed embodiment is merelyexemplary of the invention, which may be embodied in various forms.Therefore, the details disclosed herein are not to be interpreted aslimiting, but merely as a basis for teaching one skilled in the art howto make and/or use the invention.

As noted previously, the present invention results in the inactivationof organisms for the purposes of generating whole-cell therapeuticagents. Most preferably, the carbon dioxide is at or near itssupercritical pressures and temperature conditions. Thus, inactivationby the present invention may be achieved using carbon dioxide at (i) apressure from about 1000 psi to about 3500 psi and (ii) a temperaturefrom about 25° C. to about 60° C. Most preferably, carbon dioxide isheld at or near its supercritical pressure and temperature conditionsfor a time from about 20 minutes to about 12 hours. The carbon dioxideemployed in the practice of the present invention is most preferablysubstantially pure. Thus, trace amounts of other gases may be toleratedprovided that the ability of carbon dioxide to inactivate wholeorganisms is not impaired. For ease of further discussion below, theterm “supercritical carbon dioxide” will be used, but it will beunderstood that such a term is non-limiting in that carbon dioxidewithin the pressure and temperature ranges as noted immediately abovemay be employed satisfactorily in the practice of the present invention.

Therapeutic agents such as immunogenic preparations of whole organisms(or vaccines comprised thereof) prepared by the process of the presentinvention may be used for immunization and/or vaccination. The formerrequires that an immune response specific for the organism be inducedafter administration to a subject in need thereof (e.g., antibodies, Bor T lymphocytes specific for one or more antigens of the organism)while the latter provides an immune response which is prophylactic(i.e., treatment prior to infection by a pathogenic organism) ortherapeutic (i.e., treatment subsequent to infection by a pathogenicorganism). The invention involves contacting live organisms withsupercritical carbon dioxide such that they are inactivated by a factorof at least about 10⁶, at least about 10⁷, or at least about 10⁸ withoutsubstantial loss of whole cells. The resultant composition may becomprised of at least about 10⁵, at least about 10⁶, at least about 10⁷or least about 10⁸ whole organisms; alternatively, the concentration maybe at least about 10⁵, at least about 10⁶, at least about 10⁷, or leastabout 10⁸ whole organisms per milliliter. The amount of protein (e.g.,native antigen) of the organisms may be at least about 10 ng, at leastabout 100 ng, at least about 1 μg, or at least about 10 μg.

A wide range of organisms can be inactivated using the presentinvention, including for example, gram-positive bacteria, gram-negativebacteria, viruses, fungi, protozoa, and helminths. Infections which areenteric, fungal, herpesvirus, parasitic, respiratory, and vector-borne;sexually-transmitted diseases; and viral hepatitis may be treated. Giventhe low temperatures and low pressures, inactivation by supercriticalcarbon dioxide using the process of the present invention is especiallyuseful to produce whole-cell therapeutic agents (e.g., immunogens andvaccines) while maintaining the properties of thermally-labile and/orhydrolytically-labile antigens of the organisms. Spore and/or vegetativeforms resistant to phage lysis may be efficiently inactivated. Organismsdo not have to be genetically manipulated to inactivate or attenuatethem. They may be grown in culture medium or a permissive host in theindicated amount and then inactivated. Inactivation by supercriticalcarbon dioxide results in at least a 10⁶ reduction in viability orinfectivity (i.e., organisms are killed) with most of the organismshaving intact cell walls (e.g., at least about 10⁵, at least about 10⁶,at least about 10⁷, or least about 10⁸ intact whole organisms).Viability may be determined by growth in culture (e.g., number ofcolonies or plaques) or infection of susceptible hosts (e.g., morbidityor mortality of immunized subjects vs. a naive control population). Thisallows prompt development of vaccine candidates for novel pathogens andemergent diseases, which may be evaluated in animals and tried inhumans.

Organisms which may be inactivated include but are not limited to:Actinomyces spp., Bacillus anthracis, Bacillus cereus, Bordetellapertussis, Campylobacter spp., Corynebacterium diptheriae, Clostridiumbotulinum, Clostridium difficile, Clostridium perfringens, Clostridiumtetani, Corynebacterium diptheriae, Enterococcus spp., Erysipelothrixrhusiopathiae, Escherichia coli, Haemophilus influenza, Heliobacterpylori, Listeria monocytogenes, Mycoplasma pneumoniae, Neisseriameningitidis, Nocardia spp., Pseudomonas aeruginosa, Propionibacteriumacnes, Rhodococcus equi, Staphylococcus aureus, Staphylococcusepidermidis, Streptococcus agalactiae, Streptococcus pneumoniae,Streptococcus pyrogenes, and Vibrio cholerae; cytomegaloviruses, entericviruses, Epstein-Barr viruses, hepatitis viruses, herpesviruses,influenza viruses, papillomaviruses, parainfluenza viruses, poliovirus,respiratory syncytial virus, rubella virus, smallpox virus, andvaricella virus; fungi causing blastomycosis, candidiasis,coccidioidomycosis, cryptococcosis, histoplasmosis,paracoccidioidomycosis, or pythiosis; and parasites causing leprosy,malaria, or schistosomiasis. A combination of two or more differentorganisms (e.g., DPT vaccine) may be used. The transmission ofvector-borne disease may be interrupted by immunization of animalintermediaries. Animals may be immunized to produce antibodies forpassive immunization of a human subject or binding agents forimmunoassays.

A subject may be immunized (this may include vaccination in cases wherea prophylactic and/or therapeutic effect is achieved) one or multipletimes, and with or without other organisms inactivated by otherprocesses (e.g., formalin inactivation), attenuated organisms, oracellular (e.g., one or more purified antigens alone) preparations. Theamount of immunogen (or vaccine) administered to a subject in need oftherapy or prophylaxis, as well as its extent, is effective in treatmentof the subject at risk for or affected by an infectious disease. Thesubject may be any animal or human. Mammals, especially humans androdent or primate models of disease, may be treated. Thus, bothveterinary and medical methods are contemplated. The preparation may beused as primary and/or secondary immunogen (or vaccine) at suitableintervals (e.g., several weeks or months between doses).

Suitable choices in amounts and timing of doses, formulation, and routesof administration can be made with the goals of achieving a favorableresponse in the subject afflicted by an infectious disease or at riskthereof (i.e., efficacy), and avoiding undue toxicity or other harmthereto (i.e., safety). A single dose may range between 0.1 mL and 1.0mL of an immunogenic preparation containing protein of the organismsfrom about 10 ng/mL to about 10 μg/mL. The route of administration maybe enteral, mucosal, parenteral, or topical; it may be adsorbed,ingested, inhaled, or injected. For organism-specific antigens (e.g., atleast two, at least five, or at least ten different native antigens) inthe composition, labile epitopes of the antigens which are dependent ontheir conformation for immunologic activity (i.e., immunogenicity) maybe retained instead of being denatured by chemical (e.g., formalin) orphysical (e.g., heat) inactivation.

Therefore, “effective” refers to such choices that involve routinemanipulation of conditions to achieve a desired effect. It will also beunderstood that the specific dose level to be achieved for anyparticular subject may depend on a variety of factors, including age,gender, health, medical history, weight, combination with one or moreother drugs, and severity of disease. The term “treatment” of aninfectious disease refers to, inter alia, reducing or alleviating one ormore symptoms in a subject, preventing one or more symptoms fromworsening or progressing, promoting recovery or improving prognosis,and/or preventing disease in a subject who is free therefrom as well asslowing or reducing progression of existing disease. For a subject,improvement in a symptom, its worsening, regression, or progression maybe determined by an objective or subjective measure. Efficacy oftreatment may be measured as an improvement in morbidity or mortality(e.g., lengthening of survival curve for a selected population).Prophylactic methods (e.g., preventing or reducing the incidence ofrelapse) are also considered treatment. Treatment may also involvecombination with other existing modes of treatment (e.g., antibiotics,antivirals, passive immunization with antitoxin or hyperimmuneantibodies). Thus, combination treatment with one or more other drugsand one or more other medical procedures may be practiced. The amount ofimmunogen (or vaccine) which is administered to a subject is preferablyan amount that does not induce toxic or other deleterious effects whichoutweigh the advantages which result from its administration. Furtherobjectives are to reduce in number, diminish in severity, and/orotherwise relieve suffering from the symptoms of the infection ascompared to recognized standards of care.

Medicaments or pharmaceutical/diagnostic compositions may be preparedfrom the inactivated organisms. Use of preparations which furthercomprise a pharmaceutically acceptable carrier and/or other componentsuseful for delivering immunogen to a subject are known in the art.Addition of such carriers and other components (e.g., adjuvants,antibiotics, preservatives, stabilizers) to the preparation is wellwithin the level of skill in this art. Adjuvants such as aluminum orcalcium gels, bacterial toxins, cytokines, genes encoding cytokines,muramyl peptides, and saponins may be included to enhance the immuneresponse to antigen. Antibiotics include amphotericin B,chlortetracycline, gentamycin, kanamycin, neomycin, polymyxin B, andstreptomycin to prevent bacterial contamination of the compositionand/or to treat bacterial infection of the treated subject (e.g., at thesame or different time as immunization with the preparation of thepresent invention). Preservatives include benzethonium chloride,ethylenediamine-tetraacetic acid sodium, 2-phenoxyethanol, and sodiumbisulfite (formaldehyde is preferably not included). Stabilizers includealbumin, gelatin, glutamate salts, glycine, lactose, sorbitol, sucrose,and trehalose. Antibodies (e.g., hyperimmune or toxin specific) may beadministered at the same or different time as the immunogenicpreparation to provide passive immunization for a subject; suchantibodies in the form of immune globulins may be made by immunizing ananimal with the immunogenic preparation of the present invention orother preparations.

Given the existing literature and corollaries to the ghost technologyfor vaccine production, it is possible to generate some predictionsabout the properties of organisms inactivated by supercritical carbondioxide as vaccines (Table 1).

TABLE 1 Comparison of Inactivation Technologies Method of InactivationSupercritical Formalin Ghost CO₂ Speed of development + − +Non-denaturing − + + >6-Log inactivation + − + Inactivation: Gramnegative bacteria + +/− + Gram positive bacteria + − + Viruses + − +Maintains structural properties of − + + bacterium Adjuvant properties− + + No toxic chemical residues − + + Lacks antibiotic resistancegenes + − +

As noted previously, it is contemplated that contacting microorganismswith at least CO₂ at or near its supercritical pressure and temperatureconditions and an optional chemical additive sufficient to inactivatethe microorganisms and produce intact microbial cells and viruses whenassayed by various methods including transmission and scanning electronmicroscopy.

Six-log (10⁶) reductions in viability may be achieved in accordance withthe present invention by subjecting microorganisms to temperature andpressure conditions using a chemical additive-containing supercriticalcarbon dioxide as a fluid, and especially where the fluid is agitatedduring the process. Organisms may be washed after inactivation andpurified to remove the chemical additive to acceptable levels. Thepresence of trace amounts of chemical additive may be used to identify awhole organism preparation inactivated by the present invention.

The optional chemical additive used with supercritical carbon dioxidemay be comprised of peroxides and/or carboxylic acids. Preferredcarboxylic acids include alkanecarboxylic acids and alkanepercarboxylicacids, which may be optionally substituted at the alpha carbon with oneor more electron-withdrawing substituents, such as halogen, oxygen andnitrogen groups. Particularly preferred species of chemical additivesmay be comprised of hydrogen peroxide (H₂O₂), acetic acid (AcA),peracetic acid (PAA), and trifluoroacetic acid (TFA), and mixturesthereof. One particularly preferred chemical additive that may be usedis commercially available SPORECLENZ® sterilant which is a mixture ofacetic acid, hydrogen peroxide, and peracetic acid.

The chemical additive may be used in an inactivation enhancing effectiveamount of at least about 0.001 vol. % and greater, based on the totalvolume of the carbon dioxide. The amount of chemical additive will bedependent upon the particular chemical additive that is used. Thus, forexample, peracetic acid may be present in relatively small amounts ofabout 0.005 vol. % and greater, while acetic acid may need to be presentin an amount of about 1.0 vol. % and greater. Thus, a range of at leastabout 0.001 vol. % and greater, up to about 2.0 vol. % will typically beneeded in order to achieve an inactivation enhancing effect incombination with carbon dioxide.

One presently preferred embodiment of an apparatus 10 according to thepresent invention is depicted in accompanying FIGS. 1 and 2. In thisregard, it can be seen that the apparatus includes a standard compressedgas cylinder 12 containing carbon dioxide, and a standard air compressor14 used in operative association with a carbon dioxide booster 16 (e.g.,Haskel Booster AGT 7/30). Alternatively, the air compressor 14 andbooster 16 can be replaced with a single carbon dioxide compressor.

An additive cycle is also provided by means of a series of an inlet port18 which allows additive contained in reservoir 20 to be added to apressure vessel 22 through valve 24 and additive line 26. The carbondioxide is introduced to the pressure vessel 22 from header line 27 viavalve 28 and CO₂ supply line 30. A filter 32 (e.g., a 0.5 micron filter)is provided in the supply line 30 to prevent escape of material from thevessel. A pressure gauge 34 is provided downstream of CO₂ shut-off valve36 in supply header 27 to allow the pressure to be visually monitored. Acheck valve 38 is provided in the line 27 upstream of the valve 36 toprevent reverse fluid flow into the booster 16. In order to prevent anoverpressure condition existing in line 27, a pressure relief valve 9may be provided.

An outlet line 40 through valve 52 allows the pressure vessel 22 to bedepressurized. In this regard, the depressurized fluid exits the vessel22 via line 40, is filtered by filter unit 42 and then is directed toseparator 44 where filtered CO₂ gas may be exhausted via line 48, andliquid additive collected via line 50 for possible reuse. Valves 52, 54may be provided in lines 46 and 27, respectively, to allow fluidisolation of upstream components.

The reactor vessel 22 is most preferably constructed of stainless steel(e.g., 316 gauge stainless steel) and has a total internal volumesufficient to accommodate the organisms being inactivated either on alaboratory or commercial scale. For example, in laboratory studies, aninternal volume of 600 mL (e.g., approximately 8 inches long by about2.5 inches inside diameter) was deemed adequate. As is perhaps moreclearly shown in FIG. 2, the pressure vessel 22 includes a vibrator 60,a temperature control unit 62, and a mechanical stirring system mostpreferably comprised of an impeller 64 and a magnetic driver 66. Thereactor vessel 22 contains a conventional basket (not shown) which isalso preferably constructed of 316 gauge stainless steel. The basket maybe used to support one or more containers holding organisms to beinactivated as well as to protect the impeller 64 and direct the fluidin a predetermined manner.

The reactor vessel 22 may be operated at a constant pressure or undercontinual pressurization and depressurization (pressure cycling)conditions without material losses due to splashing or turbulence, andwithout contamination of pressure lines via back diffusion. The valves24, 28 and 52 allow the vessel 22 to be isolated and removed easily fromthe other components of the apparatus 10. The top 68 of the pressurevessel 22 may be removed when depressurized to allow access to thevessel's interior.

In use, the organisms to be inactivated are introduced into the interiorspace of the pressure vessel 22 along with any initial portion of liquidchemical additive from reservoir 20. The temperature control unit 62 isoperated so as to set the desired initial temperature for inactivation.The vessel 22 may then be pre-equilibrated with carbon dioxide from gascylinder 12 at atmospheric pressure, following which the magnetic driver66 is operated so as to activate the impeller 64. The pressure vessel 22may thereafter be pressurized to a desired pressure by introducingadditional carbon dioxide gas from cylinder 12 via the air compressor 14linked to booster 16.

In order to effect a pressure cycling of the vessel 22, an amount ofcarbon dioxide may be released therefrom via depressurization line bymomentarily opening valve 52 sufficient to partially reduce pressurewithin the vessel 22. Additive may be introduced into the vessel 22 forany given pressure cycle by opening valve 24 which allows liquidchemical additive to flow from reservoir 20 into inlet port 18. It willbe understood that the chemical additives may be introduced prior topressurization and/or during pressure cycling. Prior to pressurization,chemical additives may be introduced directly into the reactor vessel 22prior to sealing and/or via the additive port 18. The chemical additivesare most preferably introduced during the cycling stages by measuredaddition to the additive port 18 at ambient pressures. The port 18 issubsequently sealed and the additive chamber is pressurized so that theadditive may enter the reactor vessel 22 without altering the internalpressure. The exact mechanism of addition may be modified such that theprocess is more efficient and/or convenient.

Following additive introduction, the vessel 22 may be repressurized to adesired pressure following introduction of the liquid chemical additivetherein. Such depressurization/repressurization with introduction ofliquid chemical additive may be repeated for any number of cycles thatmay be desired. The cycle of depressurization and repressurization aswell as the introduction of the carbon dioxide and liquid chemicaladditive may be automatically controlled via a controller (not shown)which sequences the various valves discussed previously so as to achievethe desired pressure conditions and cycles.

Most preferably, periodic agitation to the contents of vessel 22 iseffected using vibrator 60 through the entire process. Intermittent orcontinuous agitation of the reactor vessel and its contents is performedby vibrating the reactor vessel during inactivation. Agitation enhancesmass transfer of the carbon dioxide and additives by eliminating voidsin the fluid such that the organism being inactivated comes into morecomplete contact with the fluid. The specific means of agitation may beadjusted to accommodate the particular apparatus employed and tooptimize the conditions for inactivation (e.g., times, temperatures,pressures, number of cycles). When processing is complete, the vessel 22is depressurized, the magnetic drive 66 is stopped thereby stopping thestirring impeller 64, and the thus inactivate whole organisms removed byopening top 68 of vessel 22.

Although the precise mechanism by which the present invention enhancesinactivation is not entirely understood at this time, it is theorizedthat, in conjunction with near-critical or supercritical carbon dioxide,the one or more optional chemical additives used in the presentinvention likely enhance inactivation by increasing the acidity of theinterior of the cell, especially in the presence of water. Moreover,chemical additives may enhance the permeability of the cell to carbondioxide, irreversibly inhibit essential cellular processes, and/orextract components required for cell viability, all of which couldpossibly contribute to enhancements in inactivation that have beenobserved.

The present invention will be further understood after carefulconsideration is given to the following Examples.

Example 1 Comparative

The effects of using an additive in accordance with the presentinvention was compared using the process described by U.S. Pat. No.6,149,864 to Dillow et al. for inactivating B. stearothermophilusspores. Specifically, the most extreme conditions as disclosed in theDillow '864 patent were evaluated (i.e., three cycles of 60° C. for twohours) and resulted in only 1.0-log inactivation (i.e., 2.3×10⁶ CFU/mLto 2.1×10⁵ CFU/mL) when no chemical additive was used (i.e., 1500 psi to3000 psi with random agitation). In contrast, 6.4-log inactivation(i.e., 2.3×10⁶ CFU/mL to undetectable) was achieved using the process ofthe present invention (i.e., 1100 psi to 3000 psi with random anddirectional agitation, and including TFA as the chemical additive). Thechemical additive was placed on a cotton ball and inserted in thechamber prior to closure. No further chemical additive was used.

Example 2 Invention

The apparatus generally depicted in FIG. 1 was employed. A sample of B.stearothermophilus spores (1 mL) of greater than 10⁶ CFU/mL was placedin 16 mm diameter test tubes in a stainless steel basket.Trifluoroacetic acid (4 mL) was transferred by syringe onto the surfaceof a cotton ball placed in the basket and water (6 mL) was placed atbottom of vessel. The basket was then loaded into the 600 mL reactorvessel. The reactor vessel was heated to 50° C. and equilibrated withCO₂ at atmospheric pressure. The stirring and agitation mechanisms wereactivated and the reactor vessel pressurized to 2000 psi for 40 minutes.The CO₂ pressure was then allowed to drop to 1100 psi at a rate of 300psi/minute. Agitation by means of vibration of the vessel was carriedout for 1 minute.

The pressurization/stirring/agitation/depressurization process wasrepeated a total of three times. After the third cycle, a series ofthree flushing cycles to remove the additive was performed bypressurizing and partial depressurizing the reactor vessel using CO₂.The stirring was stopped and the basket was removed from the reactorvessel. Any remaining CFU were counted after serial dilution andculturing of both treated and untreated controls.

Complete kill of bioindicators were achieved under different conditions.These reductions correspond to a log reduction in CFU of between 6.2 to6.9.

Example 3A Invention

A sample of B. subtilis spore and vegetative forms (1 mL) of greaterthan 10⁶ CFU/mL was placed in a 16 mm diameter test tube in a stainlesssteel basket. Acetic acid (6 mL) was transferred by syringe onto thesurface of a cotton ball placed in the basket, which was then loadedinto the 600 mL reactor vessel. The reactor vessel was heated to 50° C.and equilibrated with CO₂ at atmospheric pressure. The stirring andagitation mechanisms were activated and the reactor vessel pressurizedto 3000 psi for 40 minutes. The CO₂ pressure was then allowed to drop to1500 psi at a rate of 300 psi/minute. Agitation was carried out for 1minute.

After depressurizing the reactor vessel, more acetic acid (4 mL) wasintroduced at ambient pressure to the additive loop via port 18 (FIG.1). The loop was sealed and pressurized to 3000 psi. The reactor vesselwas the re-pressurized through the additive loop to 3000 psi such thatacetic acid was transported into the reactor vessel.

The pressurization/stirring/agitation/depressurization/chemical additioncycle was repeated a total of three times. After the third cycle, aseries of three flushing cycles to remove the additive was performed bypressurizing and depressurizing the reactor vessel using CO₂. Thestirring was stopped and the basket was removed from the reactor vessel.Any remaining CFU were counted after serial dilution and culturing ofboth treated and untreated controls.

A log reduction in CFU of between 6.0 to 6.9 was observed underdifferent conditions using the process described above.

Example 3B Invention

Example 3A was repeated except that samples containing less than 10⁶CFU/ml of B. subtilis was used. The process resulted in total kill ofthe B. subtilis present. It can therefore be extrapolated from thisexample that, had greater than 10⁶ CFU/ml of B. subtilis been presented,the process would have resulted in a corresponding 6-log reduction inCFU.

Example 3C Comparative

Example 3A was repeated except that the acetic acid was added only onceat the beginning of the process. Although a 6-log reduction in CFU wasnot observed, relatively high log reductions of between 4.5 and 4.7 wereobserved. This data suggests that multiple additions of acetic acidwould be needed in order to achieve the desired 6-log reduction in B.subtilis CFU.

Example 3D Invention

Example 3A was repeated except that pressure was maintained at aconstant 2000 psi rather than cycling. Compete kill of bioindicators wasobserved under different conditions. These log reductions in CFU rangedfrom 6.0 to 7.2.

Example 4A Invention

Example 3D was repeated except that peracetic acid was used as thechemical additive. A log reduction in CFU of between 6.5 to 7.2 wasobserved under different conditions using the process described above.

Example 4B Invention

Example 4A was repeated except that pressure was maintained at aconstant 2000 psi rather than cycling. Complete kill of bioindicatorswas observed over multiple tests with log reductions in CFU ranging from6.0 to 7.2.

Example 5 Comparative

Example 3A was repeated except that different chemical additives wereused under the conditions stated. The results appear in Table 2.

TABLE 2 Comparison of Inactivation by Various Chemical AdditivesQuantity Log Additive Temp ° C. Time (vol. %) Cycles Reduction HOCl 60 3hours 1.0 4   0-0.50 Ethanol 50-60 3 hours 1.0 4 1.2-4.0 Yeast Extract60 2 hours 1.0 3 0.37-1.1  50% Citric acid 60 2 hours 1.0 3 0.03-0.62Succinic acid 50 2 hours 1.0 3 0.25-0.29 Phosphoric acid 50 2 hours 1.03 0.18-0.25 Formic acid 50 2 hours 1.0 3 0 Malonic acid 50 2 hours 1.0 3  0-0.12

None of the additives evaluated above were effective in achieving atleast a 6-log reduction in CFU of B. stearothermophilus spores.

Example 6 Inactivation of Bacteria by Supercritical Carbon Dioxide

Maintaining the natural presentation environment for a given antigengenerally enhances the protective qualities of a given vaccine. Ghostvaccine preparations result in empty bacterial shells that are intactexcept for holes produced by the lysis gene product when viewed byscanning electron microscopy (SEM) [5, 13, 14]. In contrast, organismsinactivated by supercritical carbon dioxide have intact cell walls(i.e., no lysis holes) and are not empty (i.e., the cytosolic contentsare retained). Staphylococcus aureus and Pseudomonas aeruginosa wereinactivated by at least 6-log with supercritical carbon dioxide at 40°C. and 2973 psi to 1500 psi for a total of six cycles over 4 hours.Escherichia coli was inactivated by at least 6-log with supercriticalcarbon dioxide at 34° C. and 2973 psi to 1500 psi for a total of threecycles over 0.5 hours.

Example 7 Inactivation of B. subtilis and Protein Analysis

A sample of Bacillus subtilis (1 mL) spore and vegetative forms ofgreater than 10⁶ CFU/mL was placed into 16 mm test tubes in a stainlesssteel basket. Acetic acid (6 mL) was transferred by syringe onto thesurface of a cotton ball placed in basket, and the basket then loadedinto the 600 mL reactor vessel. The reactor vessel was heated to 50° C.and equilibrated with CO₂ at atmospheric pressure. The stirring andagitation mechanisms were activated and vessel pressurized to 3000 psifor 40 minutes. Agitation was carried out for 5 minutes. The CO₂pressure was then allowed to drop to 1500 psi at a rate of 300psi/minute.

Once the vessel was de-pressurized, 4 mL acetic acid was added atambient pressure to the additive loop. The additive loop was sealed andpressurized to 3000 psi. The vessel was then repressurized through theadditive loop to 3000 psi such that acetic acid was carried into thevessel.

The pressurization/stirring/agitation/depressurization/chemical additioncycle was repeated a total of three times. After the third cycle, aseries of three flushing cycles to remove the chemical additive wasperformed by pressurizing and de-pressurizing the reactor vessel usingCO₂. The stirring was stopped and the basket was removed from thevessel. Quantitative analysis of any remaining B. subtilis in treatedsample vs. untreated control was enumerated though serial dilutions andcolony counts. This analysis revealed that total inactivation of thebacterial preparation was achieved (i.e., at least 6-log reduction).

Inactivation was evaluated by performing SEM analysis and proteinprofiling. Specifically, it was observed that cell walls remainedintact. Moreover, extracts of B. subtilis spores both untreated andtreated with supercritical carbon dioxide were found to be virtuallyidentical. There appeared to have been no substantial loss of antigenfrom the bacteria as total protein levels were similar.

Example 8 Inactivation of S. typhimurium and Protein Analysis

A sample of Salmonella typhimurium (5 mL), the causative agent fortyphoid fever in humans, of greater than 10⁹ CFU/mL was placed into 16mm test tubes in a stainless steel basket. Water was added on a cottonball to the vessel at 1% of the total volume (1 vol. %) of the vessel.No chemical additive was placed in the vessel. The reactor vessel washeated to 35° C. and equilibrated with CO₂ at atmospheric pressure. Thestirring and agitation mechanisms were activated and vessel pressurizedto 1500 psi and held constant for 15 minutes. The vessel was thendepressurized and the contents removed for analysis. Quantitativeanalysis of any remaining Salmonella typhimurium in treated sample vs.untreated control was enumerated though serial dilutions and colonycounts. This analysis revealed that total inactivation of the bacterialpreparation was achieved after 15 minutes (i.e., 9-log reduction).

Inactivation was evaluated by performing SEM analysis and proteinprofiling. Specifically, comparative SEM analysis of untreated bacteria(FIGS. 3A-3C) and treated bacteria (FIGS. 3D-3F) revealed that cellwalls remain intact after inactivation. Moreover, protein extracts ofuntreated and treated bacteria were found to be virtually identicalafter separation by denaturing sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (FIG. 3G). In addition, thereappeared to have been no substantial loss of antigen from theinactivated bacteria as total protein levels are similar. Higherresolution protein analysis was carried out using two-dimensionalelectrophoresis (FIGS. 3H-3I). This further demonstrated that there wasno significant difference between the protein profiles of treated anduntreated bacteria. Most antigens do not appear to have been denaturedbecause native polyacrylamide gel electrophoresis during isoelectricfocusing resulted in similar migration patterns.

Example 9 Immunization with Inactivated Salmonella typhimurium

BALB/c mice will be used as subjects for immunization because they aresusceptible to S. typhimurium infection and the LD₅₀ for challenge canbe compared with and without vaccination. Immune responses will bemeasured against both homologous and heterologous whole cells andspecific for Salmonella lipopolysaccharide (LPS). An initial cohort of136 BALB/c mice will be inoculated intraperitoneally with equal doses ofimmunogenic preparations inactivated by supercritical carbon dioxide ormock inoculation (64 mice and 72 control mice).

Three different assays will be performed over the course of the 12 weekspost inoculation. Immune response assays will be performed using pooledsera collected from five different mice each week for 12 weeks as wellas pooled pre-inoculation sera from 10 mice.

Total anti-Salmonella antibodies will be measured using standardagglutination assays with homologous S. typhimurium serotype O4,5.Assays will also be performed using heterologous S. enteritidis serotypeO9 to test for any significant cross-reactivity indicative of broadprotective properties of the vaccine preparations. The results ofagglutination assays will be confirmed using a whole-cell ELISA assayfor either S. typhimurium or S. enteritidis.

Humoral responses against LPS have been shown to be a major factor inpredicting the protective properties of Salmonella vaccines. Ofparticular importance is the anti-LPS IgG2a subclass. Titers of serumIgA, IgG, IgG1, IgG2a, and IgG2b will be determined by ELISA using S.typhimurium LPS antigen. Each of the Ig classes and subclasses will beassayed using the appropriate goat anti-mouse biotinylated antibodiesand streptavidin-conjugated horseradish peroxidase. Special note will bemade of differences in Ig classes, magnitude of the immune response,duration of immune response, and cross-reactivity with heterologousserotypes.

Eight mice in each cohort will be lightly anaesthetized and thenintragastrically infected with 0.2 mL of 10 fold dilutions starting at5×10¹⁰ CFU/mL in phosphate-buffered saline (PBS). Carrier only (1× PBS)will be administered in similar fashion to mice serving as negativecontrols. Mortality will be monitored over a 30 day period usingstandard protocols and LD₅₀ calculations will be determined. Protectionwill be enumerated as the log 10 increase in LD₅₀ of immunized versuscontrol mice. A minimum number of mice will be used consistent withobtaining a statistically significant measurement of antibody responseor lack of response to an immunogen.

Thus, experiments to date support the likelihood that bacteriainactivated by supercritical carbon dioxide have potential as highquality whole-cell vaccine preparations. This is supported by theobservation that significant log reductions in CFU are achieved for awide range of bacteria while the morphology of bacteria remains intact,proteins are not significantly degraded, biodegradable polymers areunaffected, and the process is easily scaled up.

While the invention has been described in connection with what ispresently considered to be the most practical and preferred embodiments,it is to be understood that the invention is not to be limited to thedisclosed embodiments but, on the contrary, is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the present invention.

REFERENCES

-   1. Dillow, A. K., et al. (1999) Bacterial inactivation by using    near- and supercritical carbon dioxide. Proc. Natl. Acad. Sci.    U.S.A. 96:10344-10348.-   2. Duque, H., et al. (1989) Effects of formalin inactivation on    bovine herpes virus-1 glycoproteins and antibody response elicited    by formalin-inactivated vaccines in rabbits. Vaccine 7:513-520.-   3. Huter, V., et al. (2000) Improved protection against lung    colonization by Actinobacillus pleuropneumoniae ghosts:    Characterization of a genetically inactivated vaccine. J.    Biotechnol. 83:161-172.-   4. Murphy, B. R. and Walsh, E. E. (1988) Formalin-inactivated    respiratory syncytial virus vaccine induces antibodies to the fusion    glycoprotein that are deficient in fusion-inhibiting activity. J.    Clin. Microbiol. 26:1595-1597.-   5. Jalava, K., et al. (2002) Bacterial ghosts as vaccine candidates    for veterinary applications. J. Control. Release 85:17-25.-   6. Witte, A., et al. (1990) Phi X174 protein E-mediated lysis of    Escherichia coli. Biochimie 72:191-200.-   7. Eko, F. O., et al. (2000) Characterization and immunogenicity of    Vibrio cholerae ghosts expressing toxin-coregulated pili. J.    Biotechnol. 83:115-123.-   8. Eko, F. O., et al. (1994) Immunogenicity of Vibrio cholerae    ghosts following intraperitoneal immunization of mice. Vaccine    12:1330-1334.-   9. Furst-Ladani, S., et al. (1999) Bacterial cell envelopes (ghosts)    but not S-layers activate human endothelial cells (HUVECs) through    sCD14 and LBP mechanism. Vaccine 18:440-448.-   10. Haslberger, A. G., et al. (2000) Activation, stimulation and    uptake of bacterial ghosts in antigen presenting cells. J.    Biotechnol. 83:57-66.-   11. Szostak, M. P., et al. (1996) Bacterial ghosts: Non-living    candidate vaccines. J. Biotechnol. 44:161-170.-   12. Eko, F. O., et al. (1999) New strategies for combination    vaccines based on the extended recombinant bacterial ghost system.    Vaccine 17:1643-1649.-   13. Huter, V., et al. (1999) Bacterial ghosts as drug carrier and    targeting vehicles. J. Control. Release 61:51-63.-   14. Lubitz, W., et al. (1999) Extended recombinant bacterial ghost    system. J. Biotechnol. 73:261-273.-   15. Szostak, M. P., et al., Bacterial ghosts as multifunctional    vaccine particles. Behring Institute Mitteilungen, 1997(98): p.    191-6.-   16. Eko, F. O., et al. (1994) Production of Vibrio cholerae ghosts    (VCG) by expression of a cloned phage lysis gene: Potential for    vaccine development. Vaccine 12:1231-1237.-   17. Mader, H. J., et al. (1997) Endotoxicity does not limit the use    of bacterial ghosts as candidate vaccines. Vaccine 15:195-202.-   18. Lubitz, W. (2001) Bacterial ghosts as carrier and targeting    systems. Expert Opin. Biol. Ther. 1:765-771.-   19. Remaut, E., et al. (1983) Improved plasmid vectors with a    thermoinducible expression and temperature-regulated runaway    replication. Gene 22:103-113.-   20. Blasi, U., et al. (1985) Lysis of Escherichia coli by cloned phi    X174 gene E depends on its expression. J. Gen. Microbiol.    131:1107-1114.-   21. Fitzgerald, J. R., et al. (2001) Evolutionary genomics of    Staphylococcus aureus: Insights into the origin of    methicillin-resistant strains and the toxic shock syndrome epidemic.    Proc. Natl. Acad. Sci. U.S.A. 98:8821-8826.-   22. Ge, Y., et al. (2002) Extraction of natural vitamin E from wheat    germ by supercritical carbon dioxide. J. Agric. Food Chem.    50:685-689.-   23. Wu, Q. and Marshall, W. D. (2001) Approaches to the remediation    of a polychlorinated biphenyl (PCB) contaminated soil—A laboratory    study. J. Environ. Monit. 3:281-287.-   24. Fages, J., et al. (1994) Use of supercritical CO₂ for bone    delipidation. Biomaterials 15:650-656.-   25. Subramaniam, B., et al. (1997) Pharmaceutical processing with    supercritical carbon dioxide. J. Pharm. Sci. 86:885-890.-   26. Haas, G. J., et al. (1989) Inactivation of microorganisms by    carbon dioxide under pressure. J. Food Safety 2:253-265.-   27. Spilimbergo, S., et al. (2002) Microbial inactivation by    high-pressure. J. Supercritical Fluids 22:55-63.-   28. Fages, J., et al. (1998) Viral inactivation of human bone tissue    using supercritical fluid extraction. ASAIO J. 44:289-293.

The entire contents of the above cited references are herebyincorporated by reference.

While the preferred embodiments have been shown and described, it willbe understood that there is no intent to limit the invention by suchdisclosure, but rather, is intended to cover all modifications andalternate constructions falling within the spirit and scope of theinvention.

1-26. (canceled)
 27. A method of immunization, wherein said methodcomprises administering to a subject in need thereof an immunogenicpreparation of whole microorganisms, wherein said preparation iscomprised of whole microorganisms inactivated by at least a factor of10⁶ with carbon dioxide at or near its supercritical pressure andtemperature conditions and a chemical additive wherein the chemicaladditive is at least one selected from the group consisting of hydrogenperoxide, acetic acid, peracetic acid, and trifluoroacetic acid ormixture thereof.
 28. The method of claim 27, wherein said preparation isfurther comprised of an adjuvant.
 29. The method of claim 28, whereinsaid adjuvant is selected from the group consisting of aluminum orcalcium gels, bacterial toxins, cytokines, genes encoding cytokines,muramyl peptides, and saponins.
 30. The method of claim 27, wherein saidsubject is further administered one or more antibiotics and/orantibodies. 31-34. (canceled)